PROJECT SUMMARY/ABSTRACT Estrous cycle, ovulation, fertilization, and pre-implantation pregnancy are fundamental reproductive processes of clinical importance. While research has shed light on the cellular and molecular mechanisms mediating these events, much of these data are derived from static histological analysis, low-resolution visualizations, and studies of invertebrate models (e.g. sea urchin). Therefore, any conclusions regarding mammalian fertilization, which takes place deep inside the body, are extrapolated and do not necessarily represent the native state. If this technical limitation was overcome, we may gain a more complete understanding of mammalian reproduction leading to the development of better fertility treatments and Assisted Reproductive Technologies (ART). By integrating expertise in live, functional optical coherence tomography (OCT) and reproductive biology, we recently established a set of unique methods for in vivo imaging of the female mouse reproductive tract. Our approach allows for (i) live, dynamic volumetric imaging of the mouse Fallopian tube (oviduct) with micro- scale spatial resolution, (ii) depth-resolved mapping of oviduct cilia location and cilia beat frequency (CBF); and (iii) tracking of individual sperm and their motility within the oviduct. None of these measurements are currently possible with other methods, and the dynamic environment of the female reproductive tract is too complex to model. Therefore, we are in a unique position to directly visualize specific mammalian reproductive processes from an entirely new vantage point. We propose the first in vivo volumetric imaging study of mammalian fertilization. This study is taking advantage of new technological developments in OCT imaging and will allow for quantitative assessment of hormonal regulation of oviduct cilia beating and muscle contractions, and functional analysis of fertility failures in mouse models of human defects. This study will likely provide new insight on the process of mammalian fertilization in its native state and lead to a better understanding of pathologies resulting in infertility. It will also establish new functional live imaging tools, which will be a major step forward in reproductive research. Scientific Premise, Scientific Rigor, and Relevant Biological Variables: This proposal is aimed to fill a significant gap in the field of reproductive biology through highly innovative live imaging methods, which we developed. All proposed experiments are supported by strong preliminary data, which have been published in four peer-reviewed publications; one more publication is currently under review. We carefully articulated the number of experimental animals to be used, and the rationale for the choice of the models. ?Sex as a biological variable? does not apply to our study design. Extensive details and references to our published protocols are provided to ensure that preliminary and proposed experiments can be replicated in other laboratories.